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A nanobody targeting the F-actin capping protein CapG restrains breast cancer metastasis

Katrien Van Impe UGent, Jonas Bethuyne, Steven Cool, Francis Impens UGent, David Ruano-Gallego, Olivier De Wever UGent, Berlinda Vanloo UGent, Marleen Van Troys UGent, Kathleen Lambein, Ciska Boucherie, et al. (2013) BREAST CANCER RESEARCH. 15(6).
abstract
Introduction: Aberrant turnover of the actin cytoskeleton is intimately associated with cancer cell migration and invasion. Frequently however, evidence is circumstantial, and a reliable assessment of the therapeutic significance of a gene product is offset by lack of inhibitors that target biologic properties of a protein, as most conventional drugs do, instead of the corresponding gene. Proteomic studies have demonstrated overexpression of CapG, a constituent of the actin cytoskeleton, in breast cancer. Indirect evidence suggests that CapG is involved in tumor cell dissemination and metastasis. In this study, we used llama-derived CapG single-domain antibodies or nanobodies in a breast cancer metastasis model to address whether inhibition of CapG activity holds therapeutic merit. Methods: We raised single-domain antibodies (nanobodies) against human CapG and used these as intrabodies (immunomodulation) after lentiviral transduction of breast cancer cells. Functional characterization of nanobodies was performed to identify which biochemical properties of CapG are perturbed. Orthotopic and tail vein in vivo models of metastasis in nude mice were used to assess cancer cell spreading. Results: With G-actin and F-actin binding assays, we identified a CapG nanobody that binds with nanomolar affinity to the first CapG domain. Consequently, CapG interaction with actin monomers or actin filaments is blocked. Intracellular delocalization experiments demonstrated that the nanobody interacts with CapG in the cytoplasmic environment. Expression of the nanobody in breast cancer cells restrained cell migration and Matrigel invasion. Notably, the nanobody prevented formation of lung metastatic lesions in orthotopic xenograft and tail-vein models of metastasis in immunodeficient mice. We showed that CapG nanobodies can be delivered into cancer cells by using bacteria harboring a type III protein secretion system (T3SS). Conclusions: CapG inhibition strongly reduces breast cancer metastasis. A nanobody-based approach offers a fast track for gauging the therapeutic merit of drug targets. Mapping of the nanobody-CapG interface may provide a platform for rational design of pharmacologic compounds.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
metastasis, nanobody, cytoskeleton, capping protein, VHH, proteomics., MOTILITY, SEQUENCE, SECRETION, COLONIZATION, IDENTIFICATION, INVASION, NUCLEAR IMPORT, CELL-MIGRATION, GEL-ELECTROPHORESIS, ESCHERICHIA-COLI NISSLE-1917, single domain antibody
journal title
BREAST CANCER RESEARCH
Breast Cancer Res.
volume
15
issue
6
article number
R116
pages
15 pages
Web of Science type
Article
Web of Science id
000331544200012
ISSN
1465-5411
DOI
10.1186/bcr3585
language
English
UGent publication?
yes
classification
A1
copyright statement
I have retained and own the full copyright for this publication
id
4232451
handle
http://hdl.handle.net/1854/LU-4232451
date created
2014-01-14 19:51:46
date last changed
2017-05-15 09:37:27
@article{4232451,
  abstract     = {Introduction: Aberrant turnover of the actin cytoskeleton is intimately associated with cancer cell migration and invasion. Frequently however, evidence is circumstantial, and a reliable assessment of the therapeutic significance of a gene product is offset by lack of inhibitors that target biologic properties of a protein, as most conventional drugs do, instead of the corresponding gene. Proteomic studies have demonstrated overexpression of CapG, a constituent of the actin cytoskeleton, in breast cancer. Indirect evidence suggests that CapG is involved in tumor
cell dissemination and metastasis. In this study, we used llama-derived CapG single-domain antibodies or nanobodies in a breast cancer metastasis model to address whether inhibition of CapG activity holds therapeutic merit.
Methods: We raised single-domain antibodies (nanobodies) against human CapG and used these as intrabodies (immunomodulation) after lentiviral transduction of breast cancer cells. Functional characterization of nanobodies was performed to identify which biochemical properties of CapG are perturbed. Orthotopic and tail vein in vivo models of metastasis in nude mice were used to assess cancer cell spreading.
Results: With G-actin and F-actin binding assays, we identified a CapG nanobody that binds with nanomolar affinity to the first CapG domain. Consequently, CapG interaction with actin monomers or actin filaments is blocked. Intracellular delocalization experiments demonstrated that the nanobody interacts with CapG in the cytoplasmic environment. Expression of the nanobody in breast cancer cells restrained cell migration and Matrigel invasion. Notably, the nanobody prevented formation of lung metastatic lesions in orthotopic xenograft and tail-vein models of metastasis in immunodeficient mice. We showed that CapG nanobodies can be delivered into cancer cells by using bacteria harboring a type III protein secretion system (T3SS).
Conclusions: CapG inhibition strongly reduces breast cancer metastasis. A nanobody-based approach offers a fast track for gauging the therapeutic merit of drug targets. Mapping of the nanobody-CapG interface may provide a platform for rational design of pharmacologic compounds.},
  articleno    = {R116},
  author       = {Van Impe, Katrien and Bethuyne, Jonas and Cool, Steven and Impens, Francis and Ruano-Gallego, David and De Wever, Olivier and Vanloo, Berlinda and Van Troys, Marleen and Lambein, Kathleen and Boucherie, Ciska and Martens, Evelien and Zwaenepoel, Olivier and Hassanzadeh-Ghassabeh, Gholamreza  and Vandekerckhove, Jo{\"e}l and Gevaert, Kris and Fern{\'a}ndez, Luis {\'A}ngel  and Sanders, Niek and Gettemans, Jan},
  issn         = {1465-5411},
  journal      = {BREAST CANCER RESEARCH},
  keyword      = {metastasis,nanobody,cytoskeleton,capping protein,VHH,proteomics.,MOTILITY,SEQUENCE,SECRETION,COLONIZATION,IDENTIFICATION,INVASION,NUCLEAR IMPORT,CELL-MIGRATION,GEL-ELECTROPHORESIS,ESCHERICHIA-COLI NISSLE-1917,single domain antibody},
  language     = {eng},
  number       = {6},
  pages        = {15},
  title        = {A nanobody targeting the F-actin capping protein CapG restrains breast cancer metastasis},
  url          = {http://dx.doi.org/10.1186/bcr3585},
  volume       = {15},
  year         = {2013},
}

Chicago
Van Impe, Katrien, Jonas Bethuyne, Steven Cool, Francis Impens, David Ruano-Gallego, Olivier De Wever, Berlinda Vanloo, et al. 2013. “A Nanobody Targeting the F-actin Capping Protein CapG Restrains Breast Cancer Metastasis.” Breast Cancer Research 15 (6).
APA
Van Impe, Katrien, Bethuyne, J., Cool, S., Impens, F., Ruano-Gallego, D., De Wever, O., Vanloo, B., et al. (2013). A nanobody targeting the F-actin capping protein CapG restrains breast cancer metastasis. BREAST CANCER RESEARCH, 15(6).
Vancouver
1.
Van Impe K, Bethuyne J, Cool S, Impens F, Ruano-Gallego D, De Wever O, et al. A nanobody targeting the F-actin capping protein CapG restrains breast cancer metastasis. BREAST CANCER RESEARCH. 2013;15(6).
MLA
Van Impe, Katrien, Jonas Bethuyne, Steven Cool, et al. “A Nanobody Targeting the F-actin Capping Protein CapG Restrains Breast Cancer Metastasis.” BREAST CANCER RESEARCH 15.6 (2013): n. pag. Print.